Title: Facilitation of Stress-induced Phosphorylation of β-Amyloid Precursor Protein Family Members by X11-like/Mint2 Protein
Abstract: β-Amyloid precursor protein (APP) is the precursor of β-amyloid (Aβ), which is implicated in Alzheimer's disease pathogenesis. APP complements amyloid precursor-like protein 2 (APLP2), and together they play essential physiological roles. Phosphorylation at the Thr668 residue of APP (with respect to the numbering conversion for the APP 695 isoform) and the Thr736 residue of APLP2 (with respect to the numbering conversion for the APLP2 763 isoform) in their cytoplasmic domains acts as a molecular switch for their protein-protein interaction and is implicated in neural function(s) and/or Alzheimer's disease pathogenesis. Here we demonstrate that both APP and APLP2 can be phosphorylated by JNK at the Thr668 and Thr736 residues, respectively, in response to cellular stress. X11-like (X11L, also referred to as X11β and Mint2), which is a member of the mammalian LIN-10 protein family and a possible regulator of Aβ production, elevated APP and APLP2 phosphorylation probably by facilitating JNK-mediated phosphorylation, whereas other members of the family, X11 and X11L2, did not. These observations revealed an involvement of X11L in the phosphorylation of APP family proteins in cellular stress and suggest that X11L protein may be important in the physiology of APP family proteins as well as in the regulation of Aβ production. β-Amyloid precursor protein (APP) is the precursor of β-amyloid (Aβ), which is implicated in Alzheimer's disease pathogenesis. APP complements amyloid precursor-like protein 2 (APLP2), and together they play essential physiological roles. Phosphorylation at the Thr668 residue of APP (with respect to the numbering conversion for the APP 695 isoform) and the Thr736 residue of APLP2 (with respect to the numbering conversion for the APLP2 763 isoform) in their cytoplasmic domains acts as a molecular switch for their protein-protein interaction and is implicated in neural function(s) and/or Alzheimer's disease pathogenesis. Here we demonstrate that both APP and APLP2 can be phosphorylated by JNK at the Thr668 and Thr736 residues, respectively, in response to cellular stress. X11-like (X11L, also referred to as X11β and Mint2), which is a member of the mammalian LIN-10 protein family and a possible regulator of Aβ production, elevated APP and APLP2 phosphorylation probably by facilitating JNK-mediated phosphorylation, whereas other members of the family, X11 and X11L2, did not. These observations revealed an involvement of X11L in the phosphorylation of APP family proteins in cellular stress and suggest that X11L protein may be important in the physiology of APP family proteins as well as in the regulation of Aβ production. β-Amyloid precursor protein (APP) 1The abbreviations used are: APP, β-amyloid precursor protein; Aβ, β-amyloid peptide; AD, Alzheimer's disease; APLP, β-amyloid precursor-like protein; APPcyt, the cytoplasmic domain of APP; Cdk5, cyclin-dependent kinase 5; DAB1, disabled homolog 1; DLK, dual leucine zipper-bearing kinase; GST, glutathione S-transferase; JBD, JNK-binding domain; JIP, JNK-interacting protein; JNK, c-Jun NH2-terminal kinase; KLC, kinesin light chain; PI domain, phosphotyrosine interaction domain; X11L, X11-like protein; HA, hemagglutinin; HEK, human embryonic kidney; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. is a ubiquitously expressed transmembrane protein with a receptor-like structure and is the precursor of β-amyloid (Aβ) (1Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3957) Google Scholar). Aβ is a principal component of senile plaques, a characteristic pathological feature in the Alzheimer's disease (AD) brain. It is widely accepted that production, aggregation, and deposition of Aβ are closely related to AD pathogenesis; however, the detailed molecular mechanisms of this pathogenic process remain to be fully elucidated (2Hardy J. Selkoe D.J. Science. 2002; 297: 353-356Crossref PubMed Scopus (11121) Google Scholar). APP is a member of an evolutionally conserved gene family that in mammals includes amyloid precursor-like protein 1 (APLP1) and 2 (APLP2) (3Slunt H.H. Thinakaran G. Von Koch C. Lo A.C. Tanzi R.E. Sisodia S.S. J. Biol. Chem. 1994; 269: 2637-2644Abstract Full Text PDF PubMed Google Scholar, 4Wasco W. Gurubhagavatula S. Paradis M.D. Romano D.M. Sisodia S.S. Hyman B.T. Neve R.L. Tanzi R.E. Nat. Genet. 1993; 5: 95-100Crossref PubMed Scopus (322) Google Scholar). APP and its family members are physiologically important and functionally redundant because single disruption of each gene causes minor abnormalities, but combined disruption of APP and APLP2 or APLP1 and APLP2 causes lethality in early postnatal mice (5Heber S. Herms J. Gajic V. Hainfellner J. Aguzzi A. Rulicke T. Kretzschmar H. von Koch C. Sisodia S. Tremml P. Lipp H.P. Wolfer D.P. Müller U. J. Neurosci. 2000; 20: 7951-7963Crossref PubMed Google Scholar). The cytoplasmic domain of APP (APPcyt) is composed of 47 amino acid residues, and its phosphorylation at Thr668 (with respect to the numbering conversion for the APP 695 isoform) is suggested to play an important role in controlling the metabolism and physiological functioning of APP (6Suzuki T. Oishi M. Marshak D.R. Czernik A.J. Nairn A.C. Greengard P. EMBO J. 1994; 13: 1114-1122Crossref PubMed Scopus (212) Google Scholar, 7Iijima K. Ando K. 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Miller C.C. J. Neurochem. 2001; 76: 316-320Crossref PubMed Scopus (112) Google Scholar, 16Taru H. Iijima K. Hase M. Kirino Y. Yagi Y. Suzuki T. J. Biol. Chem. 2002; 277: 20070-20078Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 17Inomata H. Nakamura Y. Hayakawa A. Takata H. Suzuki T. Miyazawa K. Kitamura N. J. Biol. Chem. 2003; 278: 22946-22955Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar); JNK is a major signaling kinase in the cellular stress response and is known to be activated in neurons of AD patients (18Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar, 19Zhu X. Raina A.K. Rottkamp C.A. Aliev G. Perry G. Boux H. Smith M.A. J. Neurochem. 2001; 76: 435-441Crossref PubMed Scopus (375) Google Scholar, 20Zhu X. Castellani R.J. Takeda A. Nunomura A. Atwood C.S. Perry G. Smith M.A. Mech. Ageing Dev. 2001; 123: 39-46Crossref PubMed Scopus (285) Google Scholar). 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Commun. 1999; 255: 663-667Crossref PubMed Scopus (72) Google Scholar, 24Matsuda S. Yasukawa T. Homma Y. Ito Y. Niikura T. Hiraki T. Hirai S. Ohno S. Kita Y. Kawasumi M. Kouyama K. Yamamoto T. Kyriakis J.M. Nishimoto I. J. Neurosci. 2001; 21: 6597-6607Crossref PubMed Google Scholar, 25Trommsdorff M. Borg J.P. Margolis B. Herz J. J. Biol. Chem. 1998; 273: 33556-33560Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 26Kamal A. Stokin G.B. Yang Z. Xia C.H. Goldstein L.S. Neuron. 2001; 28: 449-459Abstract Full Text Full Text PDF Scopus (448) Google Scholar). These proteins are implicated in the metabolism and putative function(s) of APP. The X11 family proteins X11, X11-like (X11L), and X11-like 2 (X11L2) (also referred to as X11α/β/γ, Mint1/2/3, or mammalian LIN-10s) are highly homologous, especially in their carboxyl-terminal halves, which contain a phosphotyrosine interaction/phosphotyrosine-binding (PI/PTB) domain and two PDZ domains, whereas their amino-terminal regions are not very similar (21Borg J.P. Ooi J. Levy E. Margolis B. Mol. Cell. Biol. 1996; 16: 6229-6241Crossref PubMed Scopus (436) Google Scholar, 22Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 23Tanahashi H. Tabira T. Biochem. Biophys. Res. Commun. 1999; 255: 663-667Crossref PubMed Scopus (72) Google Scholar). Each of the family proteins was reported to stabilize intracellular APP and/or suppress Aβ production (22Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 27Borg J.P. Yang Y. De Taddeo-Borg M. Margolis B. Turner R.S. J. Biol. Chem. 1998; 273: 14761-14766Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 28Sastre M. Turner R.S. Levy E. J. Biol. Chem. 1998; 273: 22351-22357Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 29Ho C.S. Marinescu V. Steinhilb M.L. Gaut J.R. Turner R.S. Stuenkel E.L. J. Biol. Chem. 2002; 277: 27021-27028Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 30Araki Y. Tomita S. Yamaguchi H. Miyagi N. Sumioka A. Kirino Y. Suzuki T. J. Biol. Chem. 2003; 278: 49448-49458Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In neurons, X11 and X11L also bind Munc18, a protein essential for synaptic vesicle exocytosis (31Okamoto M. Südhof T.C. J. Biol. Chem. 1997; 272: 31459-31464Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). In addition, X11 is involved in the trafficking of a glutamate receptor by interacting with a motor protein KIF17 (32Setou M. Nakagawa T. Seog D.H. Hirokawa N. Science. 2000; 288: 1796-1802Crossref PubMed Scopus (607) Google Scholar). JIP-1 and JIP-2 are scaffold proteins within the JNK signaling cascade and function in kinesin-mediated axonal transport (33Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (589) Google Scholar, 34Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (409) Google Scholar, 35Verhey K.J. Meyer D. Deehan R. Blenis J. Schnapp B.J. Rapoport T.A. Margolis B. J. Cell Biol. 2001; 152: 959-970Crossref PubMed Scopus (499) Google Scholar). JIP-1, but not JIP-2, was also reported to retard APP metabolism and to suppress Aβ production (36Taru H. Kirino Y. Suzuki T. J. Biol. Chem. 2002; 277: 27567-27574Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Among these binding proteins, JIP-1 has been suggested to enhance the phosphorylation of APP by JNK (16Taru H. Iijima K. Hase M. Kirino Y. Yagi Y. Suzuki T. J. Biol. Chem. 2002; 277: 20070-20078Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 17Inomata H. Nakamura Y. Hayakawa A. Takata H. Suzuki T. Miyazawa K. Kitamura N. J. Biol. Chem. 2003; 278: 22946-22955Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). JIP-1 scaffolds JNK and APP to increase the phosphorylation of APP in vitro (17Inomata H. Nakamura Y. Hayakawa A. Takata H. Suzuki T. Miyazawa K. Kitamura N. J. Biol. Chem. 2003; 278: 22946-22955Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). In contrast, however, the phosphorylation level of APP is not dramatically affected by JIP-1 in cells (16Taru H. Iijima K. Hase M. Kirino Y. Yagi Y. Suzuki T. J. Biol. Chem. 2002; 277: 20070-20078Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In addition, it remains unclear whether other binding protein(s) could play a prominent role in the regulation of JNK-mediated intracellular phosphorylation. The cytoplasmic domains of APP family proteins are highly homologous, and the phosphorylation site corresponding to Thr668 of APP is conserved in APLP2 as Thr736 (with respect to the numbering conversion for the APLP2 763 isoform) (4Wasco W. Gurubhagavatula S. Paradis M.D. Romano D.M. Sisodia S.S. Hyman B.T. Neve R.L. Tanzi R.E. Nat. Genet. 1993; 5: 95-100Crossref PubMed Scopus (322) Google Scholar, 37Suzuki T. Ando K. Isohara T. Oishi M. Lim G.S. Satoh Y. Wasco W. Tanzi R.E. Nairn A.C. Greengard P. Gandy S.E. Kirino Y. Biochemistry. 1997; 36: 4643-4649Crossref PubMed Scopus (48) Google Scholar). Indeed, similar to Thr668 of APP, Thr736 of APLP2 can be phosphorylated by Cdc2 kinase (37Suzuki T. Ando K. Isohara T. Oishi M. Lim G.S. Satoh Y. Wasco W. Tanzi R.E. Nairn A.C. Greengard P. Gandy S.E. Kirino Y. Biochemistry. 1997; 36: 4643-4649Crossref PubMed Scopus (48) Google Scholar). Moreover, also similar to APP, phosphorylation of APLP2 is suggested to act as a molecular switch for binding to cytosolic proteins such as FE65 (11Ando K. Iijima K.I. Elliott J.I. Kirino Y. Suzuki T. J. Biol. Chem. 2001; 276: 40353-40361Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). However, whether the molecular mechanisms of APLP2 phosphorylation and its regulation are identical to that of APP has not been fully elucidated. In this study, therefore, we investigated the phosphorylation of APP and APLP2 by JNK and the regulation of these phosphorylation events by their binding proteins. APP and APLP2 were phosphorylated by JNK1, JNK2, and JNK3 in vitro and in cells in response to cellular stress. Furthermore, among several known binding proteins, X11L, but not X11 or X11L2, was found to markedly enhance phosphorylation of APP and APLP2. These findings further elucidate the molecular mechanisms of phosphorylation and physiological function(s) of APP and APLP2 as well as differential functions among X11 family proteins. Antibodies—Monoclonal anti-FLAG (M2, Sigma), anti-Myc (Invitrogen), anti-HA (12CA5, Roche Diagnostics), anti-GST (Upstate Biotechnology), and anti-phosphorylated JNK (G-7, Santa Cruz Biotechnology) antibodies were purchased. Polyclonal anti-active JNK and anti-phospho-c-Jun (Ser63) antibodies were purchased from Promega and Cell Signaling Technology, respectively. Polyclonal anti-APP cytoplasmic domain (G369), anti-APLP2 cytoplasmic domain (UT-424), and anti-X11L (UT-30) antibodies were as described previously (8Ando K. Oishi M. Takeda S. Iijima K. Isohara T. Nairn A.C. Kirino Y. Greengard P. Suzuki T. J. Neurosci. 1999; 19: 4421-4427Crossref PubMed Google Scholar, 22Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 38Oishi M. Nairn A.C. Czernik A.J. Lim G.S. Isohara T. Gandy S.E. Greengard P. Suzuki T. Mol. Med. 1997; 3: 111-123Crossref PubMed Google Scholar). Phosphorylation state-specific polyclonal antibodies to APP (UT-33) and anti-phospho-APP Thr668 (Cell Signaling Technology Inc.), which selectively recognize APP phosphorylated at Thr668, were as described previously (7Iijima K. Ando K. Takeda S. Satoh Y. Seki T. Itohara S. Greengard P. Kirino Y. Nairn A.C. Suzuki T. J. Neurochem. 2000; 75: 1085-1091Crossref PubMed Scopus (208) Google Scholar, 8Ando K. Oishi M. Takeda S. Iijima K. Isohara T. Nairn A.C. Kirino Y. Greengard P. Suzuki T. J. Neurosci. 1999; 19: 4421-4427Crossref PubMed Google Scholar, 37Suzuki T. Ando K. Isohara T. Oishi M. Lim G.S. Satoh Y. Wasco W. Tanzi R.E. Nairn A.C. Greengard P. Gandy S.E. Kirino Y. Biochemistry. 1997; 36: 4643-4649Crossref PubMed Scopus (48) Google Scholar). Phosphorylation state-specific polyclonal antibody to APLP2 (UT-23) was raised against a chemically phosphorylated synthetic peptide corresponding to APLP2-(732-740) (PiThr736) (37Suzuki T. Ando K. Isohara T. Oishi M. Lim G.S. Satoh Y. Wasco W. Tanzi R.E. Nairn A.C. Greengard P. Gandy S.E. Kirino Y. Biochemistry. 1997; 36: 4643-4649Crossref PubMed Scopus (48) Google Scholar). The specificity of UT-23 toward APLP2 phosphorylated at Thr736 was similar to that of UT-425 (37Suzuki T. Ando K. Isohara T. Oishi M. Lim G.S. Satoh Y. Wasco W. Tanzi R.E. Nairn A.C. Greengard P. Gandy S.E. Kirino Y. Biochemistry. 1997; 36: 4643-4649Crossref PubMed Scopus (48) Google Scholar), which was a phosphorylation state-specific antibody to APLP2 described previously. Plasmids and Peptides—The constructs pcDNA3APP695, pcDNA3-FLAGAPP695, pcDNA3-hX11L, pcDNA3-HA-JIP1b, pcDNA3-HA-JIP2, pcDNA3.1-APLP2myc/HisA, and pcDNA3.1-DLKmyc/HisA have been described previously (8Ando K. Oishi M. Takeda S. Iijima K. Isohara T. Nairn A.C. Kirino Y. Greengard P. Suzuki T. J. Neurosci. 1999; 19: 4421-4427Crossref PubMed Google Scholar, 11Ando K. Iijima K.I. Elliott J.I. Kirino Y. Suzuki T. J. Biol. Chem. 2001; 276: 40353-40361Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 16Taru H. Iijima K. Hase M. Kirino Y. Yagi Y. Suzuki T. J. Biol. Chem. 2002; 277: 20070-20078Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 22Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The cDNAs encoding JNK1α1, JNK1α2, JNK2α1, and JNK2α2 were obtained by reverse transcription-PCR from human brain mRNA (Clontech) and cloned into EcoRI/XhoI sites of pcDNA3-FLAG (36Taru H. Kirino Y. Suzuki T. J. Biol. Chem. 2002; 277: 27567-27574Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) to generate pcDNA3-FLAG-JNKs. pFlag-CMV2-JNK3 (39Ito M. Yoshioka K. Akechi M. Yamashita S. Takamatsu N. Sugiyama K. Hibi M. Nakabeppu Y. Shiba T. Yamamoto K.I. Mol. Cell. Biol. 1999; 19: 7539-7548Crossref PubMed Scopus (228) Google Scholar) was a kind gift of Dr. K. Yoshioka (Kanazawa University). The cDNAs encoding human X11, human X11L2, human FE65, mouse KLC1, mouse KLC2, and human DAB1 were obtained by reverse transcription-PCR and cloned into pcDNA3.1 tagged with HA at amino termini to generate pcDNA3.1-HA-FE65, pcDNA3.1-HA-KLC1, pcDNA3.1-HA-KLC2, and pcDNA3.1-HA-DAB1 and tagged with FLAG at amino termini to generate pcDNA3.1-FLAG-X11 and pcDNA3.1-FLAG-X11L2 (30Araki Y. Tomita S. Yamaguchi H. Miyagi N. Sumioka A. Kirino Y. Suzuki T. J. Biol. Chem. 2003; 278: 49448-49458Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The cDNAs encoding the amino-terminal region (pcDNA3hX11L-N, amino acids 1-367), the amino-terminal region attached to the PI domain (pcDNA3hX11L-N+PI, amino acids 1-555), and the carboxyl-terminal region attached to the PI domain (pcDNA3hX11L-PI+C, amino acids 368-749) of hX11L were as described previously (22Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). pcDNA3-FLAG-X11L and pcDNA3-HA-X11L were produced by PCR-mediated insertion of the FLAG and HA sequences, respectively, into the amino terminus of the full-length X11L cDNA (22Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). To construct pcDNA3-FLAGAPPΔ681-690, FLAG-APP695 deleted of amino acids 681-690 was generated by PCR using FLAG-APP695 as template and cloned into pcDNA3 using HindIII/XbaI sites. To express FLAG-APPΔ124-303, which encodes FLAG-APP695 lacking amino acids 124-303, PCR was used to produce pcDNA3-FLAG-APPΔ124-303. The cDNA encoding dual leucine zipper-bearing kinase (DLK) was amplified by PCR using pcDNA3.1-DLKmyc/HisA (16Taru H. Iijima K. Hase M. Kirino Y. Yagi Y. Suzuki T. J. Biol. Chem. 2002; 277: 20070-20078Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) as a template and cloned into pcDNA3-FLAG with EcoRI/XhoI sites to produce pcDNA3-FLAG-DLK. To construct pcDNA3-FLAG-JBD, amino acids 127-281 of mouse JIP1b (16Taru H. Iijima K. Hase M. Kirino Y. Yagi Y. Suzuki T. J. Biol. Chem. 2002; 277: 20070-20078Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) was generated by PCR and cloned into pcDNA3-FLAG (36Taru H. Kirino Y. Suzuki T. J. Biol. Chem. 2002; 277: 27567-27574Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) using EcoRI/XhoI sites. The cDNAs encoding the 47 amino acid residues of the cytoplasmic domain of APLP2 (APLP2cyt, amino acids 717-763) and the amino-terminal region of mouse c-Jun (amino acids 1-79) were also inserted into pGEX-4T-1 (Amersham Biosciences) to generate pGEX-4T-1-APLP2cyt and pGEX-4T-1-c-Jun-(1-79). pGEX-4T-1-APPcyt, pGEX-4T-1-hX11L, pGEX-4T-1-hX11L-N+PI, and pGEX-4T-1-hX11L-N were used to produce the GST fusion proteins of APPcyt and human X11L as well as the domain structures of human X11L, respectively, and have been described previously (22Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Expression and Analysis of Proteins in Cultured Cell Lines—Human embryonic kidney 293 (HEK293) cells and HEK293 cells stably expressing human APP 695 were cultured as described previously (22Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 36Taru H. Kirino Y. Suzuki T. J. Biol. Chem. 2002; 277: 27567-27574Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). For transient protein expression, HEK293 cells were transfected with plasmid expression vectors using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol. The cells were treated as indicated, collected, lysed in radioimmunoprecipitation buffer (50 mm Tris-HCl, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1% (v/v) Nonidet P-40, 0.15 m NaCl) containing 5 μg/ml chymostatin, 5 μg/ml leupeptin, 5 μg/ml pepstatin A, and 1 μm microcystin-LR, and centrifuged at 12,000 × g for 10 min at 4 °C. Protein kinase inhibitors SP600125 and SB203580 (BIOMOL Research Laboratories Inc.) were added 30 min prior to hyperosmotic treatment of cells. The resulting supernatants were subjected to immunoprecipitation with G369, UT-424, or M2 antibody as described previously (36Taru H. Kirino Y. Suzuki T. J. Biol. Chem. 2002; 277: 27567-27574Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Cell lysates or immunoprecipitated proteins were analyzed by immunoblotting with indicated antibodies using an ECL detection kit (Amersham Biosciences). For quantification, immunoblot analysis was also performed using 125I-protein A (Amersham Biosciences) with radioactivity being quantified using a Fuji BAS 1800. Co-immunoprecipitation—Co-immunoprecipitation assay was described previously (36Taru H. Kirino Y. Suzuki T. J. Biol. Chem. 2002; 277: 27567-27574Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). HEK293 cells were transfected with pcDNA3, pcDNA3-FLAGAPP695, pcDNA3-FLAGAPPΔ681-690, or pcDNA3-HA-X11L as indicated, lysed in CHAPS lysis buffer (phosphate-buffered saline containing 10 mm CHAPS, 1 mm Na3VO4, 1 mm NaF, 5 μg/ml chymostatin, 5 μg/ml leupeptin, 5 μg/ml pepstatin A, 1 μm microcystin-LR), and centrifuged at 12,000 × g for 10 min at 4 °C. The resulting supernatants were incubated with anti-FLAG M2 antibody at 4 °C for 1 h. The immunocomplex was recovered with protein G-Sepharose beads (Amersham Biosciences). Preparation of Proteins—Production and purification of recombinant GST fusion proteins were as described previously (16Taru H. Iijima K. Hase M. Kirino Y. Yagi Y. Suzuki T. J. Biol. Chem. 2002; 277: 20070-20078Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 22Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Briefly GST fusion proteins were generated in Escherichia coli BL21 containing pGEX-4T-1 cDNA constructs and purified with glutathione-Sepharose 4B (Amersham Biosciences). To prepare FLAG-tagged X11 and X11L proteins, HEK293 cells transiently expressing FLAG-X11 and FLAG-X11L were lysed and subjected to immunoprecipitation with anti-FLAG M2 affinity gel (Sigma). These proteins were eluted with elution buffer (100 μg/ml FLAG peptide (Sigma), 50 mm HEPES (pH 7.4), 100 mm NaCl). Proteins were concentrated with Microcon (Millipore) as required, and protein purity was examined by staining of gels with Coomassie Brilliant Blue R-250 following SDS-PAGE. In Vitro Phosphorylation Assay—HEK293 cells transiently expressing FLAG-JNKs were treated with 0.5 m sorbitol for 30 min at 37 °C and lysed in lysis buffer (50 mm HEPES (pH 7.5), 1% (v/v) Triton X-100, 10% (v/v) glycerol, 150 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA, 1 mm Na3VO4, 1 mm NaF, 20 mm β-glycerophosphate, 5 μg/ml chymostatin, 5 μg/ml leupeptin, 5 μg/ml pepstatin A, 1 μm microcystin-LR). FLAG-JNKs were immunoprecipitated with M2 antibody using protein G-Sepharose beads from the cell lysates. Purified GST-APPcyt or GST-APLP2cyt was incubated with the immunocomplex in kinase buffer (25 mm HEPES (pH 7.2), 10% (v/v) glycerol, 100 mm NaCl, 10 mm MgCl2, 0.1 mm Na3VO4, 5 μg/ml chymostatin, 5 μg/ml leupeptin, 5 μg/ml pepstatin A, 1 μm microcystin-LR) containing 25 μm ATP for 1 h at 30 °C. To examine the effect of X11s on APP phosphorylation by JNK, FLAG-JNK1α1 was expressed and activated as above, immunoprecipitated with anti-FLAG M2 affinity gel, and eluted with kinase buffer containing 100 μg/ml FLAG peptide. GST-APPcyt or GST-c-Jun was mixed with FLAG-X11 or FLAG-X11L proteins for 30 min and then incubated with eluted FLAG-JNK1α1 in kinase buffer containing 100 μm ATP for 1 h at 30 °C. Phosphorylation of APP and APLP2 by JNK Family Proteins—We initially examined the ability of JNK to phosphorylate APP at Thr668 and APLP2 at Thr736 (their phosphorylation sites are schematically represented in Fig. 1A). JNK proteins are expressed from three genes, Jnk1, Jnk2, and Jnk3, as low and high molecular weight splice variants (18Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar). We performed in vitro protein phosphorylation of purified APPcyt protein (Fig. 1B) or APLP2cyt protein (Fig. 1C) with activated JNK isoforms including JNK1 (α1 and α2), JNK2 (α1 and α2), and JNK3. Phosphorylation was examined by immunoblotting with antibodies that specifically recognize Thr668-phosphorylated APP or Thr736-phosphorylated APLP2. Phosphorylation of APP at Thr668 was induced by all of these JNK isoforms